Glycoprotein methods protocols - biotechnology
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Monosaccharide Composition of Mucins
Jean-Claude Michalski and Calliope Capon
1 Introduction
Mucin oligosaccharides are constructed by monosaccharide addition to form mon cores This architecture limits the number of constituent monosaccharides Monosaccharides commonly found in mucins may be divided into neutral (galactose
com-[Gal]; fucose [Fuc]), hexosamines (N-acetylgalactosamine [GalNAc];
N-acetyl-glucosamine [GlcNAc]), and acidic compounds (sialic acids [NeuAc]) Additive erogeneity comes from the possible substitution with aglycone residues such as sulfate, phosphate, or acetate groups Prior to their analysis, monosaccharides must be released from the oligosaccharide chain by acidic hydrolysis Monosaccharide composition can also be achieved on free oligosaccharide-alditols released from the native glycopro- tein by reductive alkaline treatment ( β-elimination) In this case, GalNAc is converted
het-into N-acetylgalactosaminitol (GalNAc-ol) Different methods are available for the
analysis of monosaccharides depending mainly on the amount of material available Several techniques, such as gas-liquid chromatography (GLC) or high-performance liquid chromatography (HPLC), allow both quantitative and qualitative analysis of monosaccharide mixtures Other chromatographic or electrophoretic procedures are described herein, but these only allow a rapid qualitative analysis of samples Single separated monosaccharides may be further identified by physicochemical methods such as mass spectrometry (MS) or nuclear magnetic resonance.
1.1 Release and Identification of Sialic Acids
Sialic acids constitute a family of nine-carbon carboxylated sugars found in the external position on glycan chains The diversity of sialic acids is generated by the presence of various substituents present on carbon 4, 5, 7, 8, and 9 The substituent on carbon 5 can be an amino, an acetamido, a glycolyl-amido, or a hydroxyl group and
defines the four major types of sialic acids: neuraminic acid (NeuAc), neuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and keto-deoxy-
N-acetyl-nonulosonic acid (Kdn), respectively Substituents of the hydroxyl groups present on
From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A Corfield © Humana Press Inc., Totowa, NJ
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carbons 4, 7, 8, and 9 can be acetyl, lactyl, methyl, sulfate, or phosphate, anhydro
forms can also occur (Fig 1) (1,2) Most of the substituents, largely O-acetyl groups
are quite labile during acid or alkaline hydrolysis methods generally utilized for the release of monosaccharides Consequently, the study of sialic acid must be generally considered independently of other monosaccharides The study of sialic acid modifi- cations has been attempted after release and purification by improving the methods to
avoid any destruction, and is achieved either with low concentrated acid solutions (3)
or with enzymatic hydrolysis.
Many techniques for detection and quantification of sialic acids have been described
(1) These techniques differ widely in the initial purification of sialic acids from other
biological contaminants One of the most widely used assays is the detection of free Neu5Ac and Neu5Gc acids by the thiobarbituric acid assay (TBA) Free sialic acids react with periodate under acidic conditions to produce β-formylpyruvic acid, which condenses with TBA to produce a purple chromogen ( λmax= 549 nm) The assay is sensitive to 1 nmol, but 2-deoxy-sugars interfere because they also condense with TBA to give a chromophore with a slightly lower λmax(532 nm) Powell and Hart (4)
have introduced an HPLC adaptation of the periodate–TBA assay sensitive to 2 pmol, and requiring no prior purification of released sialic acids The characterization of released sialic acids can be achieved by chromatography: thin-layer chromatography,
GLC (3), or HPLC (5–8) The last technique has higher sensitivity and resolving power We have reported the HPLC separation of sialic acid quinoxalinones (8) that
allows the detection of sialic acids at the femtomole level.
Fig 1 The sialic acids The nine-carbon backbone common to all known sialic acids may besubstituted by R1 or R2 substituents, giving a family of more than 30 different compounds
Trang 31.2 Analysis of Monosaccharides by GLC
GLC methods for identification of monosaccharides are powerful and extremely sitive Detection is usually by means of a flame ionization detector (FID), but sensitivity may be increased by coupling the gas chromatograph to an MS instrument (electron or chemical impact) Prior to analysis, monosaccharides must be released by hydrolysis of
sen-the oligosaccharides or sen-the glycoproteins and converted to a volatile derivative (9,10).
1.3 Separation of Monosaccharides by HPLC
HPLC has been widely used because of the advantages of allowing rapid and direct quantification of underivatized or derivatized samples and the ability to characterize samples through coelution with samples of known structures or through retention time
comparison Separation methods are based on anion-exchange (11), size exclusion
(12), ion suppression (13), reversed-phase (14), and, most recently, high-performance
anion-exchange chromatography (HPAEC).
HPAEC takes advantage of the weakly acidic nature of carbohydrates to give highly
selective separations at high pH using strong anion-exchange pellicular resins (15) In
HPAEC, strong alkaline solutions, usually NaOH, are used as eluent Under these
condi-tions, the hydroxyl groups of carbohydrates are converted to oxyanions with pKa values in
the range of 12–14 The anomeric hydroxyl group of the reducing sugar is more acidic than
the others but each of the hydroxyl groups is characterized by a different pKa value (16);
thus, the modification of some of the hydroxyl groups should greatly influence the elution
positions (separation of anomeric and positional isomers) (17,18) Monosaccharides
released from glycoproteins by the previously mentioned hydrolysis methods can be idly separated in less than 30 min Because their molar responses are different, a calibra- tion curve must be established for each monosaccharide When coupled with pulsed amperometric detection (PAD), HPAEC allows direct quantification of underivatized monosaccharides or carbohydrates at low picomole levels (10–50 pmol) with minimal sample preparation and purification PAD utilizes a repeating sequence of three potentials The most important potential is E1, the potential at which the carbohydrate oxidation cur- rent is measured Potential E2 is a more positive potential that oxidizes the gold electrode and completely removes the carbohydrate oxidation products The third potential, E3, reduces the oxidized surface of the gold electrode in order to allow detection during the next
rap-cycle at E1 The three potentials are applied for fixed periods referred to as t1, t2, and t3.
1.4 Electrophoretic Separation of Monosaccharides
Since the early 1990s, capillary electrophoresis has become a good alternative and rapid procedure for analytical separation of microquantities of carbohydrate com-
pounds including monosaccharides (19) Separation of native monosaccharides is
gen-erally difficult owing to the lack of ionized groups and to their low extinction coefficients, which do not allow direct ultraviolet (UV) absorbance detection Conse- quently, separation generally requires precolumn derivatization with reagents that con- tain a suitable chromophoric or fluorophoric group in order to facilitate separation and increase the sensitivity of detection As described under HPLC, the most common tagging methods are based on the reductive amination procedure, wherein the reduc-
ing end of the sugar reacts with the primary amino group of the chromophore (20).
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Different chromophores such as 2-aminopyridine (20), trisulfonic acid (ANTS) (21), ethyl-4-aminobenzoate, and 4-aminobenzonitrile (22),
8-aminonaphthalene-1,3,6-have been used for electrophoretic separation of monosaccharides.
2 Materials
2.1 Release and Identification of Sialic Acids
1 1000 mol wt cutoff dialysis tube (Bioblock Scientific, Illkirch, France)
2 Dowex AG 50 W × 8 (H+) (Bio-Rad, Hercules, CA)
3 Dowex AG 3 × 4A (HCOO–) (Bio-Rad)
4 Neuraminidase from Vibrio cholerae or Clostridium perfringens (Boehringer Mannheim,
Indianapolis, IN)
5 HPLC equipment with fluorescent detector
6 Lichrosorb RP 18 HPLC column (5-µm resin, 250 × 4.6 mm) equipped with an RT30-4Lichrosorb RP18, 7-µm guard cartridge (Merck, Darmstadt, Germany)
7 Stock solution: 2.35 mL of phosphoric acid (85%), and 28.1 g of sodium perchlorate in 1
L of distilled water
8 Working solution: water:methanol:2X buffer stock (2:3:5)
9 1,2-Diamino-4,5-methylene dioxybenzene (DMB) (Merck)
10 C18 column (250 × 4.6 mm, particle size 5 µm) (Beckman, Fullerton, CA)
11 DMB–sialic acid HPLC solvents
a Solvent A: methanol:water (7:93 v/v)
b Solvent B: acetonitrile:methanol:water (11:7:82 v/v/v)
2.2 Analysis of Monosaccharides by GLC
1 Gas-liquid chromatograph fitted with an FID
2 Magnesium turnings (Acros Organics, Geel, Belgium)
3 Sodium chloride and sulfuric acid (Sigma, St Louis, MO)
4 Meso-inositol (Sigma)
5 Silver carbonate (Sigma)
6 Acetic anhydride (Sigma)
7 Heptane (Acros Organics, Sunnyvale, CA)
8 Bis-silyltrifluoroacetamide (BSTFA) (Pierce, Austin, TX)
9 Silicone OV 101 (BP1 phase, SGE)
10 Sodium borohydride (Merck)
11 Pyridine (Merck)
12 Dichloromethane (Merck)
13 Silicone BP 70 (SGE)
14 Helium gas (Air Liquide, Paris, France)
2.3 HPLC Separation Using Amino-Bonded Silica
1 HPLC apparatus equipped with a gradient system
2 Refractive index detector
3 Kromasil–NH25µm column (250 × 4.6 mm) (Alltech, Deerfield, IL)
Trang 52.5 HPAEC-PAD
All eluents and chemical products must be of the highest purity available.
1 Gradient pump module (Dionex Bio-LC apparatus, Sunnyvale, CA)
2 A model PAD-2 detector equipped with a gold working electrode The following pulse
potentials and durations are used for detection: E1 = 0.05 V (t1= 360 ms); E2 = 0.70 V (t2
= 120 ms); E3 = –0.50 V (t3 = 300 ms) The response time is set to 3 s
3 Eluent Degas module to sparge and pressurize the eluents with helium (Dionex)
4 Postcolumn with a DQP-1 single-piston pump (Dionex).
5 CarboPac PA-1 column (4 × 250 mm) (Dionex).
6 CarboPac PA-1 guard (4 × 50 mm) (Dionex).
7 CarboPac MA-1 column (4 × 250 mm) (Dionex).
8 CarboPac MA-1 guard (4 × 50 mm) (Dionex)
9 18 MΩ deionized water (Milli-Q Plus System, Millipore, Bedford, MA)
10 NaOH 50% solution with less than 0.1% sodium carbonate (Baker, Deventer, The Netherlands)
11 Anhydrous sodium acetate (Merck)
12 Acetic acid (glacial, HPLC grade; Merck)
13 Eluents containing sodium acetate should be filtered through 0.45-µm nylon filters(Millipore) prior to use
14 Solvents for separation of neutral monosaccharides, hexosamines, and uronic acids (see
Subheading 3.3.3.2.).
a Eluent 1: Deionized water
b Eluent 2: 25 mM NaOH and 0.25 mM sodium acetate.
c Eluent 3: 200 mM NaOH and 300 mM sodium acetate.
d Eluent 4: 125 mM NaOH and 10 mM sodium acetate.
15 Solvents for HPAEC separation of sialic acids (see Subheading 3.3.3.3.).
a Eluent 1: Deionized water
b Eluent 2: 5 mM NaOAc.
c Eluent 3: 5 mM acetic acid (glacial, HPLC grade; Merck).
16 Solvents for separation of a mixture of unreduced and reduced monosaccharides (see
Subheading 3.3.3.4.).
a Eluent 1: Deionized water
b Eluent 2: 1.0 M NaOH
17 Neu5Ac and Neu5Gc acid (Sigma)
18 A mixture of sialic acids released from bovine submaxillary gland mucin (BSM) (Sigma)
(see Subheding 3.1.1.).
2.6 Electrophoretic Separation of Monosaccharides
1 Capillary zone electrophoresis apparatus fitted with a UV detector (Beckman)
2 Capillary tube (50 µm id × 65 cm) (Beckman) A part of the polyimine coating on thecapillary tube is removed by burning at a distance of 15 cm from the cathode, to allow UVdetection
3 2-Aminoacridone (AMAC) (Lambda Fluoreszentechnologie GmbH, Graz, Austria) made
up to 0.1 M in acetic acid:dimethylsulfoxide (DMSO, Acros Organics, Sunnyvale, CA)
(3:17 v/v) The solution is stored at –70°C
4 1 M sodium cyanoborohydride (Merck) in water This solution is made fresh for each
experiment
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3 Methods
3.1 Release and Identification of Sialic Acids
Figure 2 illustrates the separation of the different sialic acid species obtained after
hydrolysis of BSM The different sialic acids may be characterized according to their specific retention times Additionally, each sialic acid may be characterized by MS
analysis (8).
3.1.1 Chemical Hydrolysis of Sialic Acids
1 Suspend 1–10 mg of mucins in 5 mL of 2 M acetic acid in a Teflon-capped reaction tube.
2 Hydrolyze for 5 h at 80°C
3 Dialyze the solution for 24 h against 20 vol of water (1000 mol wt cutoff tubing)
4 Lyophilize the diffusate Direct analysis can be made at this stage
5 Further purify sialic acids as follows:
a Redissolve the dialysate in 1 mL of water
b Load the sample on a Dowex AG 50W × 8 (H+) (Bio-Rad) column (10 mL)
c Wash the column with 100 mL of water
d Lyophilize the effluent
e Resuspend the lyophilysate in 1 mL of water
Fig 2 HPLC separation of sialic acid quinoxalinones obtained after mild acid hydrolysis ofBSM Ac, acetyl; Lt, lactyl; Gc, glycolyl
Trang 7f Load the sample on a Dowex AG 3 × 4A (HCOO–) (Bio-Rad) column (1 mL).
g Wash the column successively with 7 mL of 10 mM , 7 mL of 1 M and 7 mL of 5 M
formic acid
h Pool the fractions and lyophilize
3.1.2 Enzymatic Release of Sialic Acid
1 Resuspend 1–10 mg of mucin in 2 mL of 100 mM HEPES-KOH, pH 7.0, 150 mM NaCl, 0.5 mM MgCl2, and 0.1 mM CaCl2
2 Add 200 mU/mL of V cholerae or 40 mU/mL C perfringens enzyme.
3 Introduce the solution in a dialysis tube (1000 mol wt cutoff) and dialyze against 5 mL ofthe same solvent at 37°C overnight
4 Collect the filtrate and purify the sialic acid as in Subheading 3.1.1.
3.1.3 TBA-HPLC Quantification of Sialic Acids
3.1.3.1 TBA REACTION
1 Sialic acids are released from mucins by mild acid hydrolysis as described in
Subhead-ing 3.1.1 The TBA assay is performed essentially accordSubhead-ing to Warren (23).
2 Place 40 µL of free sialic acid solution (10–100 pmol/100 µL in water) in an Eppendorf tube
3 Add 20 µL of sodium periodate (128 mg of sodium metaperiodate, 1.7 mL of phosphoricacid, and 1.3 mL of water)
4 After 20 min at room temperature, add slowly 0.1 mL of 10% sodium arsenite in 0.1 N
H2SO4,and 0.5 M Na2SO4
5 When the solution appears yellow-brown, gently vortex the tubes
6 Add 0.6 mL of 0.6% TBA (0.6 g of TBA [Sigma] in 0.5 M Na2SO4[Merck])
7 After mixing, cap the tubes and heat at 100°C for 15 min
8 Chill the tubes on ice and centrifuge before HPLC analysis
3.1.3.2 HPLC ANALYSIS (FIG 3)
1 Equilibrate the column in the working solution
2 Elute in the isocratic mode at a flow rate of 1 mL/min
3 Run UV detection at 549 nm
4 Quantify the sialic acid by integrating the surface of the sialic acid Obtain the chromophorepeak calibration curve with pure sialic acid solution (1–5 µg of sialic acid in 40 µL of water)
5 Wash the column extensively with 50% acetonitrile in water after use
3.1.4 Characterization and Quantification of Sialic Acids by HPLC
3.1.4.1 DERIVATIZATION WITH DMB
1 Heat sialic acid samples released by mild hydrolysis in 7 mM DMB, 0.75 M
β-mercaptoethanol, and 18 mM sodium hydrosulfite in 1.4 M acetic acid (100–200 µL) for2.5 h in the dark
2 Inject 10 µL of the reaction mixture on the C18 column
3.1.4.2 ELUTION BY HPLC
1 Equilibrate the column in 65% solvent A–35% solvent B
2 Elute using a linear gradient from 65% A/35% B to 100% B over 60 min followed byisocratic elution by 100% B for 10 min at a flow rate of 1 mL/min
3 Achieve on-line fluorescent detection at an emission wavelength of 448 nm and tion wavelength of 373 nm with a response time of 0.5 s
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3.2 Analysis of Monosaccharides by GLC
Complete hydrolysis of oligosaccharide chains may be obtained using concentrated acid solutions.
3.2.1 Trifluoroacetic Hydrolysis
1 Dissolve the oligosaccharide-alditol sample or the native glycoprotein in 0.5 mL of a 4 M
solution of trifluoroacetic acid (TFA) or a mixture of formic acid:water:TFA (3:2:1 v/v/v)
2 Heat at 100°C for 4 h in Teflon-capped tubes
3 After hydrolysis, remove the acid by repeated evaporation under reduced pressure ration is completed by the addition of ethanol
Evapo-3.2.2 Formic Acid–Sulfuric Acid Hydrolysis
1 Dissolve oligosaccharide-alditols or native glycoproteins in 0.5 mL of 50% aqueous mic acid and hydrolyze for 5 h at 100°C in a Teflon-capped tube
for-2 Repeat step 1 using 0.25 M aqueous sulfuric acid for 18 h at 100°C
3 Neutralize the hydrolysate with barium carbonate powder, filter, and concentrate to
dry-ness (see Note 1).
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1 Obtain anhydrous methanol by refluxing with magnesium turnings (1 h) followed by tillation in a dry all-glass apparatus
dis-2 Generate gaseous HCl by slow addition (10–20 drops/min) of sulfuric acid to 250 g ofsolid NaCl
3 Dry the hydrogen chloride gas through moisture traps containing concentrated sulfuric acid
4 Then bubble hydrogen chloride gas through anhydrous methanol for 3 to 4 h
5 Standardize the methanol–HCl reagent to 0.5 M by titration with NaOH and dilution with
anhydrous methanol (see Note 2).
3.2.3.2 METHANOLYSIS OF OLIGOSACCHARIDE OR MUCUS GLYCOPROTEIN(24)
1 Freeze-dry carefully in Teflon-capped tubes (complete dehydration of samples is themain condition of success) amounts of glycoproteins or oligosaccharides corresponding
to 10 µg of total sugar to which 1 µg of mesoinositol is added as an internal standard
2 Add 0.5 mL of methanol–HCl reagent
gly-1 Mix amounts of purified glycoproteins corresponding to 0.5 µg of total sugars with 200 µL
of 0.5 M methanol-HCl mixture for 24 h at 80°C
2 After cooling the tube, neutralize the acidic solution by adding silver carbonate to give a
pH of 6.0-7.0 as controlled with pH paper
3 Re-N-acetylate by adding 10 µL of acetic anhydride and keep overnight at room perature
tem-4 Centrifuge at 2000g for 5 min and collect the supernatant.
5 To eliminate fatty acid methyl esters, wash the methanolic phase two times with 200 µL
of heptane (remove the upper phase)
6 Dry the methanolic lower phase under a stream of nitrogen
7 Trimethylsilylate with 20 µL of BSTFA in the presence of 10 µL of pyridine for 1 h atroom temperature
8 Apply 1–5 µL of the solution of trimethylsilylated methylglycosides to GLC
3.2.4.2 GAS CHROMATOGRAPHY CONDITIONS
A typical GLC chromatogram of TMS derivatives is given in Fig 4 Neutral
monosac-charides generally provide several peaks corresponding to pyrano, furano, α, and β forms.
1 Use a FID gas chromatograph and a glass solid injector (moving needle).
2 Use a capillary column (25 m × 0.33 mm) of silicone OV 101.
3 Use carrier gas helium at a pressure of 0.5 bar.
4 Program the oven temperature from 120 to 240 °C at 2°C/min.
5 Use injector and detector temperatures of 240 and 250 °C, respectively.
Trang 11intes-3.2.5 GLC Analysis of Monosaccharides as Alditol Acetates (26) (Note 3)
3.2.5.1 HYDROLYSIS AND DERIVATIZATION
1 Hydrolyze an amount of glycoprotein corresponding to 10 µg of total sugars with 100 µL
of 4 M TFA in the presence of 2 µg of mesoinositol used as internal standard at 100°C for
4 h in glass tubes fitted with a Teflon screw cap (see Note 4).
2 After cooling, evaporate the solution and place the tube in a vacuum dessicator over P2O5
3 Reduce the liberated monosaccharides for 1 h at room temperature with 100 µL of a
solu-tion of sodium borohydride (2 mg/mL of 0.05 M ammonia solusolu-tion).
4 Destroy the excess sodium borohydride by adding of a 20% acetic acid solution untilreaching pH 4.0 Eliminate borate by codistillation with methanol (three times) in a rotaryevaporator
5 Take up the residue in 0.5 mL of water and freeze-dry
6 Peracetylate the reduced monosaccharides by adding of 50 µL of pyridine and 50 µL ofacetic anhydride, and leave overnight at room temperature
7 Remove the excess of reagent under a stream of nitrogen and take up the residue in 50 µL
of dichloromethane containing 1% of acetic anhydride
3.2.5.2 GAS CHROMATOGRAPHY CONDITIONS
A typical chromatogram is presented in Fig 5.
1 Use a gas chromatograph equipped with FID detector
2 Use a glass capillary column (12 m × 0.22 mm) of silicone BPX70
3 Use: helium at a pressure of 0.6 bar as the carrier gas
4 Program the oven temperature from 150 to 230°C and 3°C/min and then 230 to 250°C at
5°C/min
5 Use injector and detector temperatures of 240 and 250°C, respectively
6 Use an injection volume of 1 µL
3.3 Separation of Monosaccharides by HPLC
3.3.1 Separation of Native Monosaccharides by HPLC Using Bonded Silica (Kromasil-NH2)
Amino-A typical separation diagram is given in Fig 6.
1 Inject 10 µL of a monosaccharide mixture (1 mg/mL [w/v]) on to a 5-µm Kromasil-NH2column (250 × 4.6 mm)
2 Elute with acetonitrile:water (75:25 v/v) at a flow rate of 1 mL/min
3.3.2 Separation of Pyridylamino Derivatives of Monosaccharides by Reverse-Phase HPLC (27) (see Note 5)
Figure 7 presents a typical profile.
1 Hydrolyze oligosaccharides as previously described (see Subheading 3.2.).
2 Evaporate off the solvent and dissolve the residue in 100 µL of coupling reagent pared by dissolving 100 mg of 2-aminopyridine in 50 µL of acetic acid and 60 µL ofmethanol)
(pre-3 Heat the reaction mixture at 90°C for 30 min
4 Evaporate the reaction mixture under nitrogen with the addition of toluene to removeexcess reagent
5 Dissolve the pyridylamino monosaccharides in water